Protein ubiquitylation is an essential post-translational modification responsible for a diverse array of cellular processes, including protein degradation, protein trafficking, signal transduction, and the DNA damage response. Ubiquitylation is catalyzed by the concerted action of ubiquitin activating (E1), ubiquitin conjugating (E2), and ubiquitin ligase (E3) enzymes. Deubiquitylating (DUB) enzymes antagonize ubiquitylation by removing ubiquitin modifications from their substrates. Ubiquitin can be covalently conjugated to substrates in several ways: as single ubiquitin conjugated to a single site (monoubiquitylation) or multiple sites (multiple monoubiquitylation), or as a polymeric chain (polyubiquitylation). Ubiquitin can form various isopeptide linkages with itself via seven internal lysine (K) residues as well as its N-terminal methionine (M1). In addition to the homogeneous chains, it has been assumed that cells contain heterogeneous chains, such as forked or mixed chains that contain multiple types of linkages.
Accumulating evidence has suggested that the various functions of ubiquitylation are mediated by distinct chain topologies with eight different ubiquitin linkages, lengths, and complexities (FIG. 1). Of these, the linkage types are generally thought to be a critical determinant of chain function. It is widely accepted that K48-linked chains function as targeting signals for proteasomal destruction, whereas K63-linked chains are usually involved in DNA repair and the trafficking of membrane proteins. The functions of atypical chains linked through M1, K6, K11, K27, K29, or K33 are only beginning to be understood, and the roles of mixed and branched chains are unknown. Ubiquitin binding domain (UBD)-containing proteins, many of which exhibit preferences for specific ubiquitin chain types or lengths, play key roles in decoding the signals embedded in the structure of ubiquitin chains. Previous in vitro studies have shown that tetraubiquitin is the minimal recognition signal for proteasomal degradation of folded proteins. In this regard, ubiquitin ligases such as SCF and APC can build long polyubiquitin chains processively to ensure rapid degradation of their substrates. Furthermore, Rad23 and Dsk2, extrinsic ubiquitin receptors of the proteasome, preferentially bind polyubiquitin chains with four or more ubiquitins in vitro.
To understand the biological significance of different ubiquitin chain topologies, it is essential to dissect the types of ubiquitin linkages, chain complexities, and chain lengths of endogenous ubiquitylated substrates. Recent advances in mass spectrometry and antibody engineering technologies allow to determine and quantitate ubiquitin linkages in biological complex samples. In addition, the chain complexity of mixed or branched chains can be analyzed by ubiquitin linkage quantitation. By contrast, the length of substrate-attached ubiquitin chains has been analyzed only by gel mobility shift (FIG. 2). However, because most endogenous substrates have multiple ubiquitylation sites and the attached chains have intrinsically heterogeneous lengths, currently, there is no practical technique for determining the actual chain length of endogenous ubiquitylated substrates. Here a novel biochemical method for determining ubiquitin chain length is described. Using this method, the mean length of the substrate-attached polyubiquitin chains and the robustness of ubiquitin length regulation in cells were investigated.